1.2 Seed treatment and planting procedures

The seeds were pre-soaked in distilled water in beakers and left standing for 6 hours at room temperature. Facilitated aeration was carried out by intermittent agitation. The seeds were removed from the water and the excess moisture in the seeds was removed by pressing in folded filter papers. They were treated with required concentrations of 35 mM, 45 mM, 55 mM and 65 mM sodium azide using the procedure described by Klu et al. (2000). After each treatment, the seeds were washed to remove excess sodium azide under running cold tap water for about 20minutes. The sodium azide solutions were disposed according to local safety rules. Excess moisture was shaken off and the seeds spread out on blotting papers to surface dry the seeds in order to prevent the occurrence of artefacts such as unintended lesions. Two seeds were sown after soil treatment in pots.

1.3 Determination of morpho-agronomic and mutant traits

Mutants traits were observed between 3-5 days after planting include; albinism, xantha and viridis. The phenotypic traits included; plant height, stem length, leaf area, and number of leaves, days of tasseling, tasseling length, ear aspect. Plant stand and plant husk cover and mutation tolerance were rated on a scale of 1 to 5 where 1 = excellent and 5 = poor. Ear aspect was also rated visually on a scale of 1 to 5 where 1 = clean, uniform, large and well-filled ears and 5 = variable, small and partially filled ears according to the procedure described by Bello et al. (2012) and modified by Olawuyi et al. (2016).

1.4 Chromosomal studies statistical analysis

The tips of the seedling root were collected early in the morning, washed in distilled water and transferred to a specimen bottle containing 2ml Carmoy’s fixative (3:1 ethanol+ acetic acid) for 24 hours. The root tips were transferred from the fixative to another specimen bottle containing 2ml 70% ethanol for storage. During use, the root tips were hydrolyzed in IN aqueous Hydrochloric acid for 4-5 minutes in a watch glass. The hydrolyzed root tip was squashed in FLP-Orcein under No. 0 cover slip on a glass slide. The chromosome behaviours were then observed using microscope with x100 objective lens.

1.5 Statistical analysis

Data obtained were analyzed statistically using SAS generalised linear model (GLM) software version 2.5 for Analysis of Variance (ANOVA), and means were separated by Duncan Multiple Range Test (DMRT) (p≤ 0.05).

2 Results and Discussion

The result of the interactive effects of genotypes, concentration and growth stages on germination and growth characters of maize is shown in Table 2. The genotypic effect produced highly significant (p< 0.001) effects on the germination and growth traits of maize genotypes except leaf area. The concentrations of sodium azide showed highly significant mutagenic effects on percentage seed germination and height of the plants, while the growth stages had no effect on days of emergence, percentage seed germination and number of leaves. The first order interaction of Genotype x Week after planting (WAP) also produced highly significant effect (p<0.001) on the morphological traits except leaf area, while the interactive effect of genotype x concentration produced highly significant expression on plant height (Table 2). The genotypes had highly significant (p<0.001) effect on morpho-agronomic, mutagenic tolerance and yield related characters of maize. This indicates that there were variations in the interactive effects of maize genotypes (Table 3). The concentration of sodium azide had significant expression on plant stand and days to first tasseling. The result also shows that the shoot and root biomass produced highly significant effects for all the genotypes (Table 3). The better performance of EV99 QPM (Quality Protein Maize) for percentage seed germination and some morpho-agronomic traits had been previously reported by Olakojo et al. (2005), Olawuyi et al. (2014) and Olawuyi et al. (2016). The significant mean squares for the morphological and agronomic characters indicated variability among the maize genotypes by the mutagenic effect of sodium azide treatments. EV99 QPM and TZM 1441 were the most tolerant genotypes to the mutagenic effect of sodium azide. The percentage seed germination of EV99 QPM is significantly (p<0.05) higher, but different from other genotypes except TZE-WDTSTR C4, TZM 154, SAMMAZ 15 and DT-SR-COF2. Though, TZE-WDTSTR C4 and TZM 154 germinated earlier than other genotypes, while TZM 146 was the first to tassel and produced the highest cumulative number of cobs. The plant height and tasseling length of TZM 154 were significantly higher than other genotypes, while the stem height, leaf area, number of leaves and cobs, as well as root biomass of TZE-WDTSTR C4 had higher values significantly different from other genotypes (Table 4). The best agronomic traits for ear aspect and husk cover as well as shoot biomass were recorded for EV99 QPM, while TZM 146 also rated the best plant stand compared to other genotypes (Table 4). There were significant variations of morphological, agronomic and yield traits as well as mutation tolerance on the genotypes.

The result in Table 5 shows that there are no significant differences (p>0.05) in days from sowing to emergence of maize treated with varied concentrations of sodium azide. Though, untreated maize germinated earlier than the treated maize. The percentage seed germination is significantly (p<0.05) higher, but different in untreated than treated maize. The sodium azide concentrations of 35, 45 and 55 mM produced similar effects on percentage germination of seeds, but different from concentration of 65mM. It was observed that there was significant reduction in percentage seed germination of all the maize genotypes recorded at 65mM of sodium azide compared with the control (untreated). This is in accordance with concentration dose dependent inhibition in seed germination reported for different crops by Dhanavel et al. (2008) in soybean, Srivastava (2010) in wheat, Gnanamurthy and Dhanavel (2014) in cowpea and Adebola (2013) in tomato. The height of the untreated maize is higher (118.70), and not significantly different (p<0.05) from untreated ones at concentration of 35mM, but different from 45, 55 and 65 mM. On the other hand, the stem height and leaf area at 65mM were significantly higher than other concentrations and control, while number of leaves produced as well as number of days to first tasseling were similar for all concentrations. The concentration of 35 and 45mM were rated higher for ear aspect compared to other concentrations and untreated, while 45mM and 55 mM were excellently higher for husk cover. Again, the mutation tolerance was higher for untreated and concentration at 45mM. The plant stand at 45mM was fairly, but significantly rated than other treatment and control. The number of maize cobs was significantly higher and different for untreated and concentration at 65mM compared to other treatments, while the treatments at 35, 45 and 55mM were not significantly different from one another (Table 5). The tasseling length at 35mM was higher significantly than other treatments, but not different from untreated. Though, the treated maize at 35mM, 45mM and 55mM were not significantly different from one another. The concentration at 45mM produced higher significant effect on root and shoot biomass compared to other treatments and control. There were reductions in plant height, plant husk cover, plant stand, ear aspect and number of cobs in treatment concentrations compared with the control as similarly reported by Pugalandi (1992). The highest performance of 65mM observed for stem height and leaf area, as well as number of leaves, recorded best at 55mM were similar to the findings of Animasaun et al. (2014) in Arachis hypogeal. This mutagenic reduction in seed germination could be due to delay or inhibition in physiological and biological processes necessary for seed germination which include; enzyme activity (Chrispeeds and Varner, 1976), hormonal imbalance (Ananthaswamy et al., 1971) and inhibition of mitotic process (Sato and Gaul, 1967). Azide ion plays an important role in inducing mutation by interacting with enzymes and DNA in the cell. These azide anions are strong inhibitors of cytochrome oxidase which in turn inhibits oxidative phosphorylation process (Srivastava et al., 2010). In addition, it is a potent inhibitor of the proton pump36 and alters the mitochondrial membrane potential (Zhang, 2000). The mutagenic effects caused by sodium azide together may hamper ATP molecule which may reduce the germination rate and reduce the germination percentage (Srivastava et al., 2010). This could be due to low mutagenic tolerance of some maize as similarly observed by Olawuyi et al. (2016). There were variations in the effects of varied concentrations of sodium azide on morpho-agronomic and yield related traits of maize genotypes. The good performance of treated maize for 65mM for most growth and yield traits could be due to the contribution of tolerance of some maize genotypes to the mutagenic effects of sodum azide as similarly observed by Olawuyi et al. (2016). The concentration of sodium azide at 45mM was optimum for the good performance of most of the growth, agronomic and yield traits as previously reported by Adamu and Aliyu (2007).

The result of the correlation of growth, yield characters and mutation tolerance of maize genotypes to sodium azide in Table 6 showed that stem height is positively correlated with plant height and leaf area at r=0.52 and 0.57; p<0.05 respectively, while the genotype is negative, but strongly associated with plant height (r=-0.65). The number of days to first tasseling is positive and strongly related with tasseling length (r=0.88), plant stand (r=0.98), ear aspect (r=0.77), husk cover (r=0.83), number of cobs (r=0.74) and mutation tolerance (r=0.97). Also, the tasseling length is positive and strongly associated with plant stand, ear aspect, husk cover, number of cobs and mutation tolerance at r=0.86, 0.67, 0.73, 0.62 and 0.86 at p<0.01 respectively, while plant stand showed positive and strong relationship with ear aspect (r=0.79), husk cover (r=0.84), number of cobs (r=0.76) and mutation tolerance (r=0.96) (Table 6).

The Principal Component Axis (PCA) recorded from the morphological and agro-mutational traits of maize genotypes accounted for variations in Eigen values and proportion as; 3.23 (23.12%), 2.644 (18.89%), 2.033(14.53%), 1.43(10.24%), 1.06 (7.58%), 0.95 (6.79%), 0.78 (5.55%), 0.53 (3.78%), 0.40 (2.84%), 0.36 (2.60%), 0.28 (1.99%), 0.19 (1.35%), 0.10 (0.75%) (Table 7). The first PCA had the highest proportion and eigen value of 23.12% and 3,24% respectively. The ear aspect (-0.46) and husk cover (-0.41) are closely related to number of cobs (-0.40) in Prin. 1, while the stem height is closely associated with mutation tolerance, shoot and root biomass. Again, the leaf area and number of leaves are closely associated with maize grain weight. The plant height is also related to tasseling length while days of tasseling and plant stand are closely related to each other. Also, the result in Table 7 showed that plant height and stem height are highly related compared to other growth characters, while the number of leaves, days to tasseling, tasseling length, husk cover and mutation tolerance were closely related to number of cobs and maize grain weight. In Prin. 3, the number of leaves is more related to tasseling length and mutation tolerance, while the leaf area is more associated with shoot biomass and maize grain weight.

The tasseling length and maize grain weight are closely related, while the leaf area is more related to number of cobs and shoot biomass in the fourth PCA. Again, the plant height and tasseling length are closely related to ear aspect, shoot biomass and grain weight in the fifth PCA, while leaf area and tasseling length are closely associated with each other compared to husk cover, mutation tolerance and shoot biomass which are more closely related to one another in the sixth PCA (Table 7).

In Prin. 7, days of tasseling is more related to mutation tolerance, shoot and root biomass, while in Prin. 8, the leaf area was closely associated with husk cover and root biomass. The mutation tolerance in the ninth PCA had close relationship with stem height, leaf area and number of leaves, while the mutation tolerance in Prin. 10 was closely associated with number of leaves and husk cover. The days of tasseling was also more related to number of cobs and shoot biomass. The tasseling length is more associated with grain weight in Prin. 11, while husk cover is closely related with shoot biomass and number of cobs in Prin. 12. The ear aspect and husk cover were highly associated in the thirteenth PCA, while plant height and stem height are closely related in the fourteenth PCA. Growth is related to agronomic and yield traits while the agronomic traits are more associated with yield traits compared to growth characters.

The contribution of PCA and correlation matrix accounted for variation in all the traits evaluated. This is an indication of genetic variability which exists among characters as previously reported by Olawuyi et al. (2015), and Olawuyi et al. (2016) and Agbolade et al. (2016). This character could be further considered in mutation breeding.

There were variations in phenotypic expressions of sodium azide mutagen at different concentrations on the maize cultivars. The mutant traits observed on the field were; Xantha traits characterized by yellow seedling and stunted growth of TZM 146 maize treated with 45 mM of sodium azide. The death of the plant was observed within 18 days after planting (Figure 1). Albino expression was shown as white colour on leaves of TZM 146 with treatment of 55mM sodium azide. The death was also recorded in the plant 20 days after planting (Figure 2). The result in Figure 3 showed the expression of Viridis on the leaf of TZM 154 treated with 65 mM sodium azide. The colour was heterogenous with respect to green colour though, plant survived and reached maturity.

The result of the mitotic studies also revealed the behaviour of chromosomes damaged in maize induced with chemical mutagen concentrations. The abnormal metaphase was shown on EV99 QPM treated with 65mM sodium azide (Figure 4), abnormal anaphase was observed in TZM 146 at 55 mM treatment (Figure 5), while chromosomes were damaged in TZM 146 induced with 45 mM of sodium azide (Figure 6). The mutagenic effect could be due to partial or complete failure of spindle mechanism caused by mutagenicity of sodium azide mediated through the production of organic metabolite of sodium azide compound on plant chromosomes as similarly reported by Khan et al. (2009) and Gnanamurthy et al. (2014). This metabolite enters into the nucleus, interacts with DNA and creates mutation in the genome (Srivastava et al., 2010). The mutagenic effects were also morphologically shown after sowing the seeds as earlier observed by Olawuyi et al. (2016). The changes in their mitotic behavior are considered to be one of the most dependable indices to estimate the potency of mutagen (Gnanamurthy and Dhanavel, 2014).

3 Conclusion and Recommendation

There were variability responses of maize genotypes to mutagenic effects of sodium azide. Higher concentrations of sodium azide cause delay in emergence of seeds, while there was reduction in plant height and percentage germination of seeds. The number of cobs, husk cover, ear aspect, plant stand, tasseling length and days to tasseling were positive and strongly correlated with mutation tolerance with r = 0.77, 0.85, 0.76, 0.86 and 0.97 at p< 0.01 respectively. The first Principal Component Axis (Prin. 1) highly contributed to the variations in morphological, yield and mutation tolerance traits, while the growth characters are more related to agronomic and yield related traits. The mutation tolerance is more related to growth and agronomic traits compared to yield traits. The use of calculated and optimum amount of pesticide constituting sodium azide should also be taken into consideration as higher concentration dose could be harmful and mutagenic to the plants.

The findings from the cytological studies provided information on the mutagenic response of maize genotypes to sodium azide, and thus, provide greater possibilities for selection of desired characters and resistant genotypes. TZE-WDTSTR C4, TZM 154 and TZM 154 were observed to be early and maturing genotypes. Also, the characters that showed variability should be considered in future mutagenic breeding. Therefore, the selection of EV99 QPM and TZM 1441 which were the most tolerant genotypes to the chemical mutagen should be considered in mutagenic breeding programs so as to facilitate maize improvement.

Authors' contributions

Dr Olawuyi, O.J. designed the research study, managed the literature searches, wrote the protocol, interpreted the data and produced the initial draft. Mr Okoli, S.O. anchored the field study, anchored the data and performed preliminary data analysis. The two authors read and approved the final manuscript.

Acknowledgements

The authors are grateful to National Centre for Genetic Resources and Biotechnology (NACGRAB), Ibadan, Nigeria for providing the maize genotypes.Special thanks also go to the Department of Botany, University of Ibadan, Nigeria for providing the necessary facilities.

References

Adamu, A. K. and H. Aliyu, 2007. Morphological effect of NaN₃ on tomato (L.esculentum). Science World J. 2(4):9-12

Adebola, M. O. 2013. Mutagenic effects of sodium azide (NaN3) on morphological characteristics of tomato (lycopersicum esculentum). Intl J.Res. Pub., 2(4): 1563-2251

Agbolade, J. O., O. J. Olawuyi, O. B. Bello, O. D. Oluseye and R. J. Komolafe, 2016. Genetic diversity and correlated response to selection of grain and associated characters in maize (Zea mays L.). Intl. Res. J. Appl. Basic. Sci., 13(1): 56-61

Ajala, S. O. 2005. A summary of the final Report Doubling Maize Production in Nigeria in two Years; A Presidential Initiative (Nov2005-Nov2007)

Akande, S. O. 1994. Comparative Cost and Return in Maize Production in Nigeria. Nigeria Institute for Social and Economic Research (NISER) individual Research Project Report, Ibadan: NISER

Ananthaswamy, H.M., V. K. Vakil and A.Shrinivas, 1971. Biological and physiological changes in gamma irradiated wheat during germination. Radiat. Bot., 11: 1-12

https://doi.org/10.1016/S0033-7560(71)91257-9

Animasaun, D. A., S. Oyedeji, M. A. Azeez and A. O. Onasanya, 2014. Evaluation of the Vegetative and Yield Performances of Groundnut (Arachis hypogaea) Varieties Samnut 10 and Samnut 20 Treated With Sodium Azide Intl J. Sci. Res., 4(3): 2250-3153

Arulbalachandran, D., L.Mullainathan and S. Velu, 2009. Screening of mutants in black gram (Vigna mungo (L.) Hepper) with effect of DES and COH in M₂ generation. J. Phytol., 1: 213-218

Bello O. B., S. Y. Abdulmaliq, S. A. Ige, J.  Mahamoo, F. Oluleye, M. A. Azeez, M. S. Afolabi, 2012. Evaluation of Early and Late/Intermediate Maize Varieties for Grain Yield Potential and Adaptation to a Southern Guinea Savanna Agro-ecology of Nigeria. Intl J. Plant Res. 2(2): 14-21

https://doi.org/10.5923/j.plant.20120202.03

Bello, O. B. and G. Olaoye, 2009. Combining ability for maize grain yield and other biological systems. Mut. Res., 197: 313–323

Bertagne-Sagnard, B., G. Fouilloux and Y. Chupeau, 1996. Induced albino mutations as a tool for genetic analysis and cell biology in flax (Linum usitatssimum). J. Exp. Bot., 47: 189–194

https://doi.org/10.1093/jxb/47.2.189

Broertjes, C. and A.M. van Harten, 1988. Applied Mutation Breeding for Vegetatively Propagated Crops. Elsevier, New York, USA., ISBN-13: 9780444427861, Pages: 345

Bulk., R.W., H.J.M. Loffler, W.H. Lindhout and M. Koornneef, 1990. Somaclonal variation in tomato: Effect of explant source and a comparison with chemical mutagenesis. Theor. Appl. Genet., 80: 817-825

https://doi.org/10.1007/BF00224199

Chrispeeds, M. J. and J. E. Varner, 1976. Gibberelic acid induced synthesis and release of amylase and ribonuclease by isolated barley aleurons layers. Plant Physio., 42: 346-406

Dhanavel, D., P. Pavadai, L. Mullainathan, D. Mohana, G. Raju, M. Girija and C. Thilagavathi, 2008. Effectiveness and efficiency of chemical mutagens in cowpea (Vigna unguiculata (L.) Walp.). Afr. J. Biotechol., 7: 4116-4117

Gnanamurthy, S. and D. Dhanavel, 2014. Effect of EMS on induced morphological mutants and chromosomal variation in Cowpea (Vigna unguiculata (L.) Walp). ILNS 17:33-43

https://doi.org/10.18052/www.scipress.com/ILNS.22.33

Khan, M. S., P. A. Zaidi and M. Oves, 2009. Role of plant growth promoting rhizobacteria in the remediation of metal contaminated soils.  Environmental Chemistry Newsletter. Pp 1-9
https://doi.org/10.1007/978-1-4020-9654-9_15

Kleinhofs, A., W. M. Owais and R. A. Nilan, 1978. Azide. Mutat. Res., 55: 165-195

https://doi.org/10.1016/0165-1110(78)90003-9

Klu, G. Y. P., H. M. Amoaley, D. Bansa, and E. K Kumanya, 2000. Plant Genet. Resour. Newsl., 124: 13-16

Kulthe, M.P. and V. S. Kothekar, 2011. Effects of sodium azide on yield parameters of chickpea (Cicer arietinum L.). J.  Phytol., 3: 39-42

Mensah, J. K. and B. Obadoni, 2007. Effects of sodium azide on yield parameters of groundnut ( Arachis hypogaea L.). Afr. J. Biotechnol., 6: 668-671

Morris, M. L. 1998. Overview of the world maize economy. In: M. L. Morris (ed.). Maize Seed Industries in Developing Countries. Lynne Rienner Publishers, Inc. and CIMMYT, Int. pp. 13-34

Mostafa, G.G. 2011. Effect of sodium azide on the growth and variability induction in Helianthus annuus L. Intl. J. Plant Breed. Genet., 5: 76-85

https://doi.org/10.3923/ijpbg.2011.76.85

OECD. 2003. Consensus document on the biology of Zea mays subsp. mays (Maize). Report No. 27, Environment Directorate; Organisation for Economic Co-operation and Development Paris, France

Ojo, S.O. 2003. Productivity and technical efficiency of poultry egg production in Nigeria, Int. J. Poult. Sci., 2(6): 459-464. https://doi.org/10.3923/ijps.2003.459.464

Olakojo S. A. 2004. Evaluations of maize inbreed lines for tolerance to Striga lutea in Southern Guinea Savannah Ecology. J. Food Agric. Environ., 2(2):256- 259

Olakojo S. A., J. O. S. Kogbe, J. E. Iken and A.  M. Daramola, 2005. Yield and disease evaluation of some improved maize (Zea mays L) varieties in Southwestern Nigeria. Trop. Subtrop. Agroeco., 5:51-55

Olawuyi, O. J., A. C.  Odebode, B. J. Babalola, E. T. Afolayan and C. P. Onu, 2014. Potentials of arbuscular mycorrhiza fungus in tolerating drought of miaze (Zea mays L.). Am. J. Plant Sci., 5: 779-786

https://doi.org/10.4236/ajps.2014.56092

Olawuyi, O. J., A. C. Odebode and S. A. Olakojo, 2013. Genotype × treatment × concentration interaction and character association of maize (Zea mays L) under arbuscular mycorrhiza fungi and Striga lutea Lour. In Proceedings of the 37th Annual Conference of the Genetics Society of Nigeria (GSN), Lafia, 20-24 October, pp. 210-219

Olawuyi, O. J., O. B. Bello and A. O. Abioye, 2016. Mutagenic effects of Ultraviolet Radiation on growth and agronomic characters in maize cultivars. Mol. Plant Breed., 7(1): 1-10

Olawuyi, O. J., O. B. Bello, C. V. Ntube and A. O. Akanmu, 2015. Progress from selection of some maize cultivars’ response to drought in the derived savanna of Nigeria. Agrivita J. Agric. Sci., 37(1): 8-17

https://doi.org/10.17503/Agrivita-2015-37-1-p008-017

Olawuyi, O.J., A.C. Odebode, A. Alfar-Abdullahi, S. A. Olakojo and A. I. Adesoye, 2010. Performance of maize genotypes and arbuscular mycorrhizal fungi in Samara district of South West region of Doha-Qatar. Nig. J. Mycol., 3(1): 86-100

Pugalendi, N. 1992. Investigation on induced mutagenetics in Sesamum indicum L. M.Sc. (Ag.) Thesis, Annamalai University, Annamalai Nagar, Tamil Nadu

Salim, K., A. Fahad and A. Firoz, 2009. Sodium Azide: a Chemical Mutagen for Enhancement of Agronomic Traits of Crop Plants. Environ. We. Int. J. Sci. Tech.., 4: 1-21

Sato, M., and H. Gaul, 1967. Effect of ethyle methane sulphonate on the fertility of barley.  Radiat. Bot., 7: 7-15

https://doi.org/10.1016/0033-7560(67)90028-2

Srivastava, P., S. Marker, P. Pandey and D. K. Tiwari, 2010. Mutagenic Effects of Sodium Azide on the Growth Yield Characteristics in Wheat (Triticum aestivum L.em.Thell.). Asian J.Plant Sci., 10: 190-201

USDA. 2005. Germplasm Resources Information Network - (GRIN) [Online Database]. United States Department of Agriculture, Agricultural Research Service, Beltsville, available online at http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl 14-3

Wongpiyasatid, A., S. Chotechuen, P. Hormchan, S. Ngampongsai and W. Promcham, 2000. Induced mutations in mungbean breeding: Regional yield trial of mungbean mutant lines. Kasetsart J. Nat. Sci. Res., 34: 443-449

Zhang, B. H. 2000. Regulation of plant growth regulators on cotton somatic embryogenesis and plant regeneration. Biochem., 39: 1567